1. Field of the Invention
The present invention relates to a cell-permeable p53 recombinant protein in which a macromolecule transduction domain (MTD) is fused to the tumor suppressor p53, a polynucleotide encoding the same, a recombinant expression vector for producing the same, and a pharmaceutical composition of the treatment of cancer caused by p53 deficiency or by the loss of p53 function, comprising the same.
2. Description of the Related Art
The tumor suppressor p53 is a potent transcription factor that plays a central role in large networks of proteins that integrate a variety of signal transduction pathways including cell cycle regulation, apoptosis initiation, DNA repair, senescence and other anticancer functions. In light of the finding that approximately 50% of primary tumors have mutations in the p53 gene, with the observation of mutant p53 regulators in the remainder, p53 can be seen to be responsible for the effective suppression of human tumors. In fact, the repair of p53 in p53-mutant tumor cells was found to lead to apoptosis of the cells.
In normal cells, p53 levels are kept low because p53 proteins are inactivated by association with MDM2 (Murine double minute 2) and because they are degraded rapidly on synthesis. In response to various stresses including DNA damage, the expression of the p53 protein increases and becomes activated to prevent malignant transformation by inducing cell cycle arrest and apoptosis. The cell cycle is the series of events that take place in a cell leading to its division and duplication and consists of four distinct phases: the G1 phase, the S phase for DNA replication, the G2 phase, and the M phase for cell division. The cell cycle is regulated at various checkpoints therein. p21 is activated by the transcription factor p53 and halts the cell cycle in the G1 phase. When activated by p53, p21 binds to and inactivates cyclin-CDK (cyclin-dependent kinase) complexes.
MDM2 (Murine double minute 2) is a representative p53 inhibitor that binds to the p53 transactivation domain to prevent p53 from inducing the expression of target genes. Further, acting as a ubiquitin ligase targeting p53, MDM2 induces the ubiquitination of p53 and mediates the degradation of p53 by the 26S proteasome pathway. The p53 transcription factor induced in damaged cells activates the transcription of MDM2 while MDM2 promotes the degradation of p53, which, in turn, increases the expression of MDM2. As such, there is an autoregulatory feedback loop between p53 and MDM2.
Mutant or inactivated p53 is found in most human tumor cells. p53-mediated signal transduction pathways exhibit very complicated tissue specificity. In some cell types such as fibroblasts and epithelial cells, p53 acts to induce permanent or transient cell cycle arrest in the G1 and G2 phases of the cell cycle to prevent the cells from proliferating or growing when the DNA is in a state of damage and repair. Human p53 is 393 amino acids long and has a modular structure comprised of four main functional domains. Of them, the DNA binding domain (DBD, residues 102-292) is known to play a core role in the functions of p53. Approximately 90% of the known mutations that deactivate p53 in cases of cancer occur in this domain. As for the regulation and activation of p53, it is achieved mainly by posttranslational modifications including ATM (ataxia-telangiectasia mutated)/ATR (ataxia telangiectasia and Rad3 related)-mediated phosphorylation at the Ser/Thr residues, the up-regulation of p53 transcriptional activity by histone acetylase, ubiquitination at carboxyl-terminal lysine residues, and sumoylation.
One of the most effective approaches to the molecular therapy of cancer is to rehabilitate the function of wild-type p53. For this, a lot of effort has been conventionally focused on gene delivery so that wild-type p53 will be expressed in tumor cells having mutant p53 or on the finding of small molecules that can activate p53 in tumor cells. Despite all attempts, the suggestions made thus far remain at the stage of mere possibility, with the appearance of inadequate physical and chemical properties, and thus have not yet culminated in characteristic cancer therapeutics.
To overcome the problems encountered with conventional attempts, p53 was imparted with cell permeability by fusion into a macromolecule transduction domain (MTD), which facilitates the transduction of macromolecules into cells. As a result, recombinant p53, called cell permeable-p53 (CP-p53), has been developed for anti-cancer therapeutics.
The macromolecule transduction domain (MTD) developed by the present inventors is capable of cell-to-cell delivery so that it allows the systemic transduction of macromolecules upon administration upon any route. Hence, CP-p53, when fused to the MTD, can be effectively transduced into cells, which might certainly result in the development of protein-based bio-drugs that could cure diseases caused by p53 deficiency or by the loss of p53 function. Under the conviction that the overexpression within cells or direct delivery into cells of p53 could suppress the formation and growth of a tumor, the present inventors have put a lot of effort into developing novel anticancer agents using MTD.
Synthetic compounds or natural small molecules can be transduced into cells, but macromolecules such as proteins, peptides, nucleic acids, etc. cannot due to their large sizes. It is difficult for macromolecules with a molecular weight of 500 or more to pass through a plasma membrane, that is, the lipid bilayer structures, of viable cells. To overcome this problem, macromolecule intracellular transduction technology (MITT) was developed (Korean Patent Application No. 10-2009-7017564, U.S. patent application Ser. No. 12/524,935, Canadian Patent Application No. 2,676,797, Chinese Patent Application No. 00880003468.9, Australian Patent Application No. 2008211854, Indian Patent Application No. 5079/CHENP/2009, EU Patent Application No. 08712219.8, Japanese Patent Application No. not yet assigned (priority based on USPP: 60/887,060)). MITT facilitates the transverse of therapeutic macromolecules across membranes into cells, thus making it possible to develop peptides, proteins and genes themselves as bio-drugs, which is impossible using conventional techniques. According to MITT, macromolecules are fused to hydrophobic macromolecule transduction domains and other various intracellular vehicles, expressed and purified as recombinant proteins. When administered into the body, the recombinant proteins are transduced into cells and delivered to intracellular target sites where the macromolecules functionally operate. As described, MTD allows the conveyance into cells of various molecules which are incapable of permeating into cells, such as peptides, proteins, DNA, RNA, synthetic molecules, etc.
Culminating in the present invention, intensive and thorough research into the use of p53 in the treatment of cancer, conducted by the present inventors, resulted in the finding that when imparted with cell permeability by fusion into MTD, the tumor suppressor p53 can be effectively translocated into cell nuclei in vivo as well as in vitro and that the p53 recombinant protein can be used as an anticancer agent for treating cancers caused by p53 deficiency or by the loss of p53 function.